G-proteins serve as molecular switches responding to a variety of extracellular cues to relay intracellular responses. Dysregulated G-protein signaling leads to pathophysiologies in numerous organ systems. The current project will utilize phage display peptides that bind to specific nucleotide-bound states of G-alpha subunits in a combined approach of structural biology, biochemistry, and live cell imaging to more clearly elucidate the spatiotemporal dynamics of G-protein signaling pathways. Aim 1: To delineate the structural determinants of nucleotide-selective binding of phage peptides to cognate G-alpha subunits using x-ray crystallography Aim 2: To determine the effects of phage peptides on the nucleotide cycle of G-protein subunits as potential direct modulators of G-protein regulation using in vitro biochemical approaches Aim 3: To determine the spatiotemporal dynamics of G-protein activation using phage peptides as in vivo biosensors that incorporate environmentally-sensitive, solvatochromatic dyes with live cell imaging techniques.